Genetic primary microcephaly (PM) is a defect in early brain development leading to congenital microcephaly, mostly recessively inherited, and mild-to-moderate intellectual disability. PM has been largely elucidated, thanks to exome and genome sequencing. However, radial microbrain, the most severe form of genetic PM or micrencephaly described in the 1980s, which leads to early lethality or very severe intellectual handicap, remains without a molecular diagnosis. We sought to identify the cause of radial microbrain by analyzing the genotype of children/adults and fetuses with an extremely small brain.

We searched for individuals with the smallest head circumference among patients with a confirmed diagnosis of PM included in 2 French and European observational studies coordinated at the Robert Debré Children's Hospital in Paris. Their neurodevelopment and brain imaging were analyzed, as well as next-generation sequencing for a panel of microcephaly genes or exome sequencing. Neuropathologic and immunohistologic analyses of extremely severe microcephalic fetal brains and stage-matched controls were performed. A nonparametric test and Mann-Whitney post-test were used to compare the cortical thickness between groups.

We identified 5 individuals (4 female patients, 7 years 10 months-19 years) with a particularly small brain among a series of 50, all suffering from a severe neurodevelopmental disorder with no ability to communicate verbally and, in 3 of them, no ability to walk. Genetic analysis revealed in all individuals the presence of the same homozygous variant c.2953A>G (p.R985G) in the RTTN gene (ROTATIN). The same variant was found in 2 fetuses whose neuropathologic evaluation showed a major reduction in the thickness of the ventricular zone and neuronal heterotopias. The cortical plate was reduced by 70% compared with controls, irrespective of the region considered. Immunostaining with vimentin showed a 50% loss of radial glial columns, characteristic of radial microbrain.

Our data show that the homozygous c.2953A>G substitution in RTTN is a recurrent variant responsible for radial microbrain, the most severe form of primary microcephaly. Our combined neurologic, imaging, and histopathologic approaches provide a better understanding of the severity of this condition and its prognosis.